Nucleic acid encoding a transdominant negative retroviral integrase

ABSTRACT

A method of using a transdominant negative integrase gene to make at least one cell resistant to a retroviral infection which includes retroviral infections resulting from HIV; a method for introducing a transdominant negative integrase gene into at least one mammalian cell to make said cell resistant to a retroviral infection as well as vectors, cells, and methods of constructing same useful in the afore-mentioned methods; a method of treating AIDS comprising administering to a patient an effective amount of a transdominant negative integrase gene alone or combined with agents useful for gene therapy inhibition of HIV, antiviral agents, or interleukin-2; and pharmaceutical delivery methods which include a transdominant negative integrase gene alone or combined with agents useful for gene therapy inhibition of HIV, antiviral agents, or interleukin-2.

This application is a divisional of application Ser. No. 08/841,179,filed Apr. 29, 1997, U.S. Pat. No. 5,908,923, which is a divisional ofapplication Ser. No. 08/286,578, filed Aug. 5, 1994, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a medical method of treatment. Inparticular, the present invention concerns the use of a transdominantnegative integrase gene to make at least one mammalian cell resistant toa retroviral infection, to methods for their production, topharmaceutical delivery methods which include these genes, and topharmaceutical methods of treatment. More particularly, the noveltransdominant negative integrase gene alone or combined with anothergene that confers protection from human immunedeficiency virus (HIV)such as, for example, a transdominant negative rev gene and/or aribozyme that cleaves HIV ribonucleic acid (RNA) is useful in treatingacquired immunedeficiency syndrome (AIDS).

AIDS results from HIV infection which depletes CD4⁺T cells. Currently,there is no effective treatment for AIDS. One of the most attractive andleast exploited targets for the therapy of AIDS is the viral integrase(Brown P. O., “Integration of Retroviral DNA”. In: Current Topics inMicrobiology of Immunology, 157:19-48 (1990)). The life cycle ofretroviruses is dependent on integration into the host chromosome. ForHIV, integration is necessary for viral replication (LaFemina R. L., etal., Journal of Virology, 66:7414-7419 (1992) and Sakai H., et al.,Journal of Virology, 67:1169-1174 (1993)). This process is mediated byintegrase, a viral protein. One approach to inhibition of the essentialintegration process is to express some form of integrase that will blockthe integrase function of the incoming HIV. This is based on the conceptof “pathogen-derived resistance” disclosed by Sanford J. C. and JohnstonS. A., J. Theor. Biol., 113:395-405 (1985). Pathogen-derived resistanceis based on the strategy that expression of certain genes from pathogensinhibit replication of such pathogens. Various examples of this concepthave been disclosed including: retroviral envelope genes (Robinson H.L., et al., Journal of Virology, 40:745-751 (1981)); coat protein genesof plant viruses (Wilson T. M. A., Proc. Natl. Acad. Sci. USA,90:3134-3141 (1993)); envelope glycoprotein genes of herpes viruses(Petrovskis E. A., et al., Journal of Virology, 62:2196-2199 (1988));transdominant negative HIV rev (Malim M. H., et al., Cell, 58:205-214(1989)); over-expression of HIV tar sequences as “decoys” (Sullenger B.A., et al., Cell, 63:601-608 (1990)); and transdominant negative mutantsof HIV gag. In the case of HIV, this concept of pathogen-derivedresistance was later termed “intracellular immunization” (Baltimore D.,Nature, 335:395-396 (1988)).

However, prior to the present invention, there was no suggestion thatretroviral integrase could be made to exert a transdominant negativephenotype. In fact, since integrase enters a cell with the virion and ispresumed to remain part of the preintegration complex, it was regardedby many as an unlikely candidate for being amenable to interference by atransdominant negative mutant. In a recent review on gene therapy ofAIDS (Yu M., et al., Gene Therapy, 1:13-26 (1994)), many possibilitieswere discussed with no mention of integrase as a target. Additionally,prior to the present invention, there was no good technology forexploring the possibility of a transdominant negative integrase.Retroviral integrases are made as part of polyprotein precursors (the“gag-pol precursor”) in infected cells. There is no natural expressionof a retroviral integrase in a mammalian cell without many other viralproteins that are part of the precursor. Although there is a report ofexpression of Rous sarcoma virus integrase alone (Mumm S. R., et al.,Virology, 189:500-510 (1992)), expression of HIV integrase has beenproblematic. This problem has been attributed to a rev-responsiveelement within the integrase gene (Cochrane, et al., J. Virol.,65:5305-5313 (1991)). Holler T. P., et al., Gene, 136:323-328 (1993)reported the synthesis of genes coding for wild-type (“NdeI”) and aninactive mutant (“D116N:) integrase for expression in E. coli.

Thus, an object of the present invention is the expression of HIVintegrase in mammalian cells. Efficient expression of HIV integrase wasachieved in the present invention by employing a synthetic gene forexpression in mammalian cells (Seq ID No.: 1). That this was successfulwas an unpredictable and surprising result, since the synthetic geneused was synthesized to optimize codon usage for the bacterium E. coli.Bacterial genes could have sequences recognized by mammalian cells assplice sites or methylation sites for inactivation of the gene, makingthe successful expression of a bacterial gene in mammalian cells highlyunpredictable.

SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to a method of using atransdominant negative integrase gene to make at least one cellresistant to a retroviral infection.

In a preferred embodiment of the first aspect of the invention, theretroviral infection is selected from the group consisting of: HIV; HTLVI; HTLV II; equine infections anemia virus; bovine leukemia virus;murine retrovirus; and avian leukosis virus, and the cell is selectedfrom the group consisting of a mammalian cell and an avian cell.

In a most preferred embodiment of the first aspect of the invention, theretroviral infection is HIV and the cell is a human cell.

In a second aspect, the present invention is directed to a method forintroducing a transdominant negative integrase gene into at least onemammalian cell to make said cell resistant to a retroviral infection,comprising introducing the DNA into at least one mammalian cell by aprocess of delivery selected from the group consisting of:

a) use of calcium phosphate co-precipitation;

b) in a complex of cationic liposomes:

c) electroporation;

d) receptor mediated endocytosis;

e) naked DNA;

f) transduction by a viral vector; and

g) particle-mediated gene transfer (Cheng L., et al., Proc. Natl. Acad.Sci. USA, 90:4455-4459 (1993) and Yang N-S., Critical Reviews inBiotechnology, 12:335-356 (1992)).

In a preferred embodiment of the second aspect of the invention, thetransdominant negative integrase gene is HIV integrase and the mammaliancell is a human cell.

In a most preferred embodiment of the second aspect of the invention,the human cell is selected from the group consisting of a CD4⁺Tlymphocyte, a monocyte, and a hematopoietic progenitor cell, and theviral vector is selected from the group consisting of: retrovirus;adenovirus; adeno-associated virus; and herpes virus.

In a third aspect, the present invention is directed to a method forintroducing a transdominant negative integrase gene into at least onemammalian cell to make said cell resistant to a retroviral infectioncomprising inserting said transdominant negative integrase gene into avector and expressing transdominant negative integrase in said at leastone mammalian cell.

In a preferred embodiment of the third aspect of the invention, thetransdominant negative integrase gene is HIV integrase; the vector isselected from the group consisting of: pRSV/IN-NdeI and pRSV/IN-D116N;and the mammalian cell is 293-CD4.

In a more preferred embodiment of the third aspect of the invention, thetransdominant negative integrase gene is HIV integrase; the vector ispRSV/IN-NdeI; and the mammalian cell is 293-CD4.

In a most preferred embodiment of the third aspect of the invention, thetransdominant negative integrase gene is HIV integrase; the vector ispRSV/IN-NdeI; and the mammalian cell is a human cell selected from thegroup consisting of a CD4⁺T lymphocyte, a monocyte, and a hematopoieticprogenitor cell.

In a fourth aspect, the present invention is directed to a method ofconstructing the pRSV/IN-NdeI vector comprising inserting the HIVintegrase gene into the pRcRSV vector to afford the pRSV/IN-NdeI vector.

In a fifth aspect, the present invention is directed to a vector forproduction of HIV transdominant negative integrase comprising a DNAsequence encoding HIV integrase.

In a preferred embodiment of the fifth aspect of the invention, thevector is selected from the group consisting of pRSV/IN-NdeI andpRSV/IN-D116N.

In another preferred embodiment of the fifth aspect of the invention,the vector is pRSV/IN-NdeI.

In a more preferred embodiment of the fifth aspect of the invention, theDNA sequence contains a substantial number of codons different from thenatural codons, preferably 10 or more codons different from the naturalcodons.

In a most preferred embodiment of the fifth aspect of the invention, theDNA sequence is Seq ID No.: 1 or a DNA sequence containing 10 or lesscondons different from Seq ID No.: 1 or a fragment thereof that encodesat least 150 amino acids; and expression is carried out in a eukaryoticcell, preferably a mammalian cell.

In a sixth aspect, the present invention is directed to the syntheticgene of Seq ID No.: 1 or a synthetic gene having a DNA sequencecontaining 10 or less codons different from Seq. ID. No.: 1 or afragment thereof that encodes at least 150 amino acids.

In a seventh aspect, the present invention is directed to a highlytransfectable cell line which is transduced to express human CD4.

In a most preferred embodiment of the seventh aspect of the invention,the highly transfectable cell line is 293-CD4.

In an eighth aspect, the present invention is directed to a method ofconstructing a 293-CD4 cell comprising inserting the CD4 coding sequenceinto a vector and transfecting the vector into 293 cells.

In a most preferred embodiment of the eighth aspect of the invention,the vector is RSV-CD4.

In a ninth aspect, the present invention is directed to a method ofpreparing a retroviral integrase comprising inserting a syntheticintegrase gene into a vector and expressing said vector in a mammaliancell.

In another preferred embodiment of the ninth aspect of the invention,the retroviral integrase is HIV integrase.

In a more preferred embodiment of the ninth aspect of the invention, thesynthetic integrase gene contains a substantial number of codonsdifferent from the natural codons, preferably 10 or more codonsdifferent from the natural codons.

In a most preferred embodiment of the ninth aspect of the invention, thesynthetic integrase gene is Seq ID No.: 1 or a DNA sequence containing10 or less codons different from Seq ID No: 1 or fragment thereof thatencodes at least 150 amino acids.

A transdominant negative integrase gene could be delivered (by aretrovirus or other gene delivery method) to CD4⁺T cells from anHIV-infected patient ex vivo, then re-introduced into the patient. Thiswould give the patient a population of T cells that would be resistantto infection by HIV. Additionally, a transdominant negative integrasegene could be delivered (by a retrovirus or other gene delivery method)to hematopoietic progenitor cells. Such cells can be derived from bonemarrow or from peripheral blood after stimulation by a cytokine such as,for example, G-CSF. These transduced cells are then reintroduced intothe patient. This would give a population of hematopoietic cells,including CD4⁺T cells that would be resistant to HIV.

Use of a transdominant negative integrase gene as a method of treatmentof AIDS is based on the following scientific rationale:

1. Integrase is an essential protein for HIV infection. Genetic analysisof HIV integrase mutations (LaFemina R. L., et al., Journal of Virology,66:7414-7419 (1992) and Sakai H., et al., Journal of Virology,67:1169-1174 (1993)) shows that integrase is essential for HIV infectionto proceed. Additional genetic analysis by Shin, C-G., et al., Journalof Virology, 68:1633-1642 (1994) shows that changes in integrase canhave pleiotropic effects on a variety of steps in the HIV life cycle.Although we do not yet know the step in the HIV life cycle where thedominant negative integrase is having its effect, it is clearly avulnerable target for interfering with replication of virus.

2. Cell culture results where HIV replication is blocked is the acceptedcriterion by the AIDS research community and regulatory authorities forclinical testing. This includes cell culture effect of transdominantnegative mutants of retroviral proteins, as exemplified by the approvalof clinical testing of rev M10 (Nabel G., et al., Human Gene Therapy,5:79-92 (1994)).

3. Transdominant negative genes active in cell culture providepathogen-derived resistance at the level of an intact multicellularorganism. Examples include retroviral envelope genes and plant viruscoat protein genes (Robinson H. L., et al., Journal of Virology,40:745-751 (1981);

Wilson T. M. A., Proc. Natl. Acad. Sci. USA, 90:3134-3141 (1993)).

Thus, in a tenth aspect, the present invention is directed to a methodof treating AIDS in a patient comprising administering to said patient atherapeutically effective amount of a transdominant negative integrasegene.

The transdominant negative integrase gene might advantageously be usedin combination with any of a variety of other agents useful for genetherapy inhibition of HIV (Yu M., et al., Gene Therapy, 1:13-26 (1994)and Yamada O., et al., Gene Therapy, 1:38-45 (1994)).

Thus, in a eleventh aspect, the present invention is directed to amethod of treating AIDS in a patient comprising administering to saidpatient a therapeutically effective amount of a transdominant negativeintegrase gene in combination with one or more agents selected from thegroup consisting of:

a) a transdominant negative gene such as, for example, a transdominantnegative rev gene, a transdominant negative tat gene: a transdominantnegative gag gene, a transdominant negative env gene, a transdominantnegative vpx gene, and the like;

b) a soluble(s) CD4 gene such as, for example, a sCD4 gene, a sCD4-KDELgene, and the like;

c) an intracellular antibody;

d) an interferon-inducible gene such as, for example, a RBP9-27 gene andthe like;

e) a RNA decoy gene such as, for example, HIV-1 TAR, HIV-1 RRE, and thelike;

f) an antisense RNA; and

g) a ribozyme such as, for example, a hammerhead ribozyme, a hairpinribozyme, and the like.

Preferably, the transdominant negative integrase gene mightadvantageously be used in combination with other transdominant negativeHIV genes, for example, a rev gene (e.g., the rev M10 of Malim M. H., etal., Cell, 58:205-214 (1989)) and/or ribozymes that cleave HIV RNAs(Yamada O., et al., Gene Therapy, 1:38-45 (1994)). This would enable agreater degree of efficacy than would be achieved by any one gene.

Thus, in a twelfth aspect, the present invention is directed to a methodof treating AIDS in a patient comprising administering to said patient atherapeutically effective amount of a transdominant negative integrasegene in combination with a transdominant negative rev gene.

In a most preferred embodiment of the twelfth aspect of the invention,the transdominant negative rev gene is rev M10.

In a thirteenth aspect, the present invention is directed to a method oftreating AIDS in a patient comprising administering to said patient atherapeutically effective amount of a transdominant negative integrasegene in combination with a ribozyme that cleaves HIV RNA.

In a fourteenth aspect, the present invention is directed to a method oftreating AIDS in a patient comprising administering to said patient atherapeutically effective amount of a transdominant negative integrasegene in combination with a transdominant negative rev gene and aribozyme that cleaves HIV RNA.

In a fifteenth aspect, the present invention is directed to apharmaceutical delivery method adapted for administration to a patientin an effective amount of an agent for treating a retroviral infectioncomprising a transdominant negative integrase gene and a suitable viralor nonviral delivery system.

In a preferred embodiment of the fifteenth aspect of the invention, thepharmaceutical delivery method is adapted for ex vivo or in vivodelivery.

In a most preferred embodiment of the fifteenth aspect of the invention,the pharmaceutical delivery method is directed to therapeutic orprophylactic administration.

In a sixteenth aspect, the present invention is directed to a method oftreating AIDS in a patient comprising administering to said patient atherapeutically effective amount of a transdominant negative integrasegene in combination with an antiviral agent.

In a preferred embodiment of the sixteenth aspect of the invention, theantiviral agent is selected from the group consisting of a nucleoside ora nonnucleoside reverse transcriptase inhibitor, a HIV proteaseinhibitor, and a tat inhibitor.

In a most preferred embodiment of the sixteenth aspect of the invention,the reverse transcriptase inhibitor is selected from the groupconsisting of azidothymidine, dideoxyinosine, dideoxycytosine, and d4T.

In a seventeenth aspect, the present invention is directed to a methodof treating AIDS in a patient comprising administering to said patient atherapeutically effective amount of a transdominant negative integrasegene in combination with interleukin-2.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by the following nonlimiting exampleswhich refer to the accompanying FIG. 1, short particulars of which aregiven below.

FIG. 1 shows the amino acid (Seq. ID NO: 2) and nucleotide sequence(Seq. ID No: 1) of the synthetic gene which encodes the integraseprotein reengineered with translation initiation sequences of mammaliancells.

DETAILED DESCRIPTION OF THE INVENTION

The term “transdominant” means that the effect of the gene isoperational when the gene is expressed from some genetic element notnecessarily linked to the virus.

The term “negative” means that the gene reduces replication of theretrovirus.

The term “transdominant negative” means a gene that can inhibitreplication of a retrovirus without necessarily being genetically linkedto the retrovirus.

The term “transdominant negative integrase gene” includes an intactretroviral integrase gene, fragments thereof, and both active andcatalytically inactive mutants thereof.

The term “transfected gene” means a gene introduced into a cell by someexogenous means, such that a gene is added that the cell did not receivefrom the germ line of the animal from which it was derived.

The term “transient expression” means the expression of a transfectedgene that is temporary, usually lasting only a few days.

The term “stable expression” means the expression of a transfected genewhere the expression is sustained for weeks.

The term “HIV” includes HIV-1 and HIV-2.

The term “mammal” includes humans.

The present invention includes 293-CD4 cells. Human cells expressing theCD4 gene are generally infectable with HIV, since CD4 is the receptorused by HIV for cell entry. The literature teaches that candidatetransdominant negative genes are tested by expressing the transfectedgene in a stable cell line, and then testing that stable cell line forits ability to resist infection by HIV (see Yu M., et al., Gene Therapy,1:13-26 (1994) and references cited therein). Making such a stable cellline expressing the candidate gene is laborious, taking more than 1month of selection and screening cells for expression.

A faster method would be to use so-called “transient expression” of thecandidate gene, where cells are tested within 24-48 hours aftertransfection. However, in most cell lines, transient expression isinefficient. Inefficiency refers to the percentage of cells in thepopulation that take up and express the gene being introduced. In mostcell lines, fewer than 10% and often around 1% of the cells in apopulation take up and express the transiently transfected gene. Thiswould clearly be unacceptable for evaluating candidate transdominantnegative genes, since if over 90% of a cell population remainsuntransfected and fully susceptible to HIV infection, a small percentageof resistant cells would not be experimentally detectable.

The present invention solves this problem by introducing the gene forCD4 in an expression vector into the transformed human kidney cell line293 (available from American Type Culture Collection). This isexemplified in Example 1. The result is a cell line that can betransiently transfected at a frequency of greater than 50%, and byvirtue of its CD4 expression, is infectable with HIV. This enables atest of candidate dominant negative genes by transfection and infectiona day later.

This is both surprising and unexpected since it could not be predictedthat such a cell line would be useful for testing candidate dominantnegative genes. Thus, prior to the present disclosure, it was not knownthat 293-CD4 cells would be infectable with HIV or that the 293-CD4cells would retain the highly efficient transient expression capabilityof the 293 cell parent. Moreover, prior to the present disclosure, itwas not known that the kinetics of transient expression (e.g.,expression of the transfected gene declines after about 2 days) wouldallow significant protection from an HIV challenge or that protection of293-CD4 cells from HIV challenge by transient expression would bepredictive for protection of a naturally susceptible cell population.

Therefore, the present invention affords a cell line and protocol thatcan be used to discover the transdominant negative effect of integrasegenes; and that will be broadly useful in research on dominant negativemutants of HIV proteins, as well as other kinds of protective genes suchas ribozymes.

Also, the present invention incudes a vector for production of HIVtransdominant negative integrase in a mammalian cell incorporating asynthetic integrase gene, e.g., a DNA sequence which contains asubstantial number of codons different from the natural codons such as,for example, 10 or more codons different from the natural codons or afragment thereof that encodes at least 150 amino acids. Preferably, theDNA sequence is Seq ID No.: 1 or a DNA sequence containing 10 or lesscodons different from Seq ID No.: 1, or a fragment thereof that encodesat least 150 amino acids.

Additionally, the present invention includes transdominant negativeretroviral integrase genes and methods to use these transdominantnegative integrase genes to render cells resistant to retroviralinfection. This would include making a population of cells in an HIVinfected person resistant to HIV via delivery of such genes.

The transdominant negative retroviral integrase gene can be introducedinto cells by any of the many methods known for introducing DNA intocells, either transiently or permanently. The methods for introducingDNA into cells include calcium phosphate co-precipitation, cationicliposomes, electroporation, receptor mediated endocytosis,particle-mediated gene transfer, or for some cell types, naked DNA canbe used. The transdominant negative integrase genes can also beintroduced by any of the well-known viral vectors, includingretroviruses, adenovirus, adeno-associated virus, and herpes viruses.For some applications, e.g., making an animal resistant to a retrovirus,the transdominant negative integrase might be introduced into the germline of an animal by the methods for making transgenic animals(including pronuclear microinjection, embryonic stem cells, and othertechnologies known in the art). Thus, the transdominant negativeintegrase gene of the present invention can be introduced into cells byconventional gene transfer technology known to those skilled in the art.

In addition, the transdominant negative integrase gene could be combinedwith any of the variety of other approaches for gene therapy inhibitionof HIV (Yu M., et al., Gene Therapy, 1:13-26 (1994) and Yamada O., etal., Gene Therapy:1:38-45 (1994)).

Thus, the transdominant negative integrase gene may be combined with oneor more agents selected from the group consisting of:

a) a transdominant negative gene such as, for example, a transdominantnegative rev gene, a transdominant negative tat gene, a transdominantnegative gag gene, a transdominant negative env gene, a transdominantnegative vpx gene, and the like;

b) a soluble(s) CD4 gene such as, for example, a sCD4 gene, a sCD4-KDELgene, and the like;

c) an intracellular antibody;

d) an interferon-inducible gene such as, for example, a RBP9-27 gene andthe like;

e) a RNA decoy gene such as, for example, HIV-1 TAR, HIV-1 RRE, and thelike;

f) an antisense RNA; and

g) a ribozyme such as, for example, a hammerhead ribozyme, a hairpinribozyme, and the like.

Preferably, a transdominant negative integrase gene of the presentinvention may be combined with a transdominant negative rev gene suchas, for example, a transdominant negative rev M10 gene as described byMalim M. H., et al., Cell, 58:205-214 (1989) and/or a ribozyme thatcleaves HIV RNAs. Ribozymes and methods for their preparation have beendisclosed in U.S. Pat Nos. 4,987,071, 5,037,746, 5,116,742, 5,093,246,and 5,180,818 which are hereby incorporated by reference.

Additionally, specific anti-HIV ribozymes have been disclosed inInternational Published Patent Applications WO 9401549-A1, WO9324133-A1, WO 933569-A1, WO 9207065-A1, WO 9201806-A, WO 9110453-A, WO9103162-A, WO 9013641-A; European Published Patent Application EP360257-A, and U.S. Pat. No. 5,144,019 which are hereby incorporated byreference.

Optimal treatment of a patient receiving dominant negative integrasegene therapy will often involve coadministration with a chemicalantiviral drug or interleukin-2. Currently approved drugs that can becombined with integrase gene therapy are azidothymidine, dideoxyinosine,dideoxycytosine, or d4T. The invention envisions combination with futureantivirals in the classes of nucleoside and non-nucleoside reversetranscriptase inhibitors, HIV protease inhibitors, and tat inhibitors.

A suitable pharmaceutical delivery method for the dominant negativeintegrase genes is either by ex vivo or in vivo delivery. In the case ofex vivo delivery, CD4⁺T cells, monocytes, or hematopoietic progenitorcells, are removed by plasmapheresis from either the patient or asuitable donor. The dominant negative integrase gene is then introducedinto these cells by transduction with a retroviral vector or bymicroprojectiles (Nabel G., et al., Human Gene Therapy, 5:79-92 (1994)).Alternatively, the genes could be introduced via adeno-associated virus(e.g., Zhou S. Z., et al., J. Exp. Med., 179:1867-1875 (1994)) orliposomes. The transduced cells, either with or without selection forsurvival of transduced cells, are then administered to the patient to betreated. Usually a dose of 1×10⁷ to 1×10¹¹ transduced T cells, or from 1to 1×10⁶ transduced hematopoietic progenitor cells are administered percourse of treatment. The patient may be given repeat courses oftreatment periodically as required to maintain a suitable level oftransduced CD4⁺T cells, usually with 3 months to 3 years betweentreatments.

For in vivo delivery, a suitable viral or nonviral delivery system isused to administer the dominant negative integrase gene to the patient.This administration may be intravenous. The formulation could be, forexample, using cationic liposomes (Philip R., et al., J. Biol. Chem.,268:16087-16090 (1993)), where from 10 μg to 10 mg of a vectorexpressing the dominant negative integrase is delivered. For in vivoadministration, it will usually be preferred to use a vector that willdirect tissue-specific gene expression, e.g., the promoter of the humanCD4 gene.

The following nonlimiting examples illustrate the inventor's preferredmethods for preparing a transdominant negative integrase gene of thepresent invention.

EXAMPLE 1

Construction of 293-CD4 Cells

293 cells are a transformed human cell line (available from the AmericanType Culture Collection) that are particularly useful for efficienttransient expression of transfected genes. The gene for human CD4 (thereceptor for HIV) is introduced into these cells to make themsusceptible to HIV infection.

Step A Preparation of RSV CD4 Expression Vector

A 3.0 kb fragment containing 1.8 kg of CD4 coding sequence is removedfrom the T4-pMV7 plasmid (Maddon P. J., et al., Cell, 47:333-348 (1986))using EcoRI. The ends are made blunt by Klenow polymerase. The pRSV PAPplasmid (Lin, et al., Biotechniques, 3:344-348, 350-351 (1991)) is cutwith HindIII and XbaI to remove the insert, and the ends made blunt withKlenow polymerase. The CD4 fragment is then ligated into the plasmidbackbone.

Step B Preparation of 293-CD4 Cells

A calcium phosphate transfection is performed to introduce the RSV-CD4vector into 293 cells, using the method described in Sambrook J., etal., Molecular Cloning. A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989, 16.30-16.40. The cells are transfected at 20%confluence, washed with Dulbecco's Modified Eagle's Medium (DMEM) plus10% fetal calf serum 24 hours posttransfection, and selected in G418 at0.5 mg/mL 48 hours posttransfection. Following G418 selection,individual clones are isolated and screened by fluorescence activatedcell sourcing (FACS) for CD4 using a monoclonal antibody to CD4.

EXAMPLE 2

Construction of a Vector Expressing HIV Integrase in Mammalian CellsUsing a Synthetic Gene

The synthetic genes coding for wild-type (“NdeI”) and an inactive mutant(“D116N”) integrase (IN) had previously been cloned into the E. coliexpression vector pKK223 (Holler T. P., et al., Gene, 136:323-328(1993)). Constructs for the NdeI and D116N gene are done in parallel;all manipulations described are done for both genes. The first uniquerestriction site in the synthetic gene is a ClaI site at nucleotide 17(relative to the ATG). The plasmid pKK223/NY5IN-NdeI (or D 116N) isdigested with ClaI and dephosphorylated using bacterial alkalinephosphatase (BRL). Synthetic oligonucleotides ALM 1 (5′-CCAAGCTGG GCCACCATG GCC TTC CTG GAC GGT AT-3′) (Seq ID No: 3) and its complement ALM 2(5′- CGAT ACC GTC CAG GAA GGC CAT GGT GGC CCA AGC TTGG-3′) (Seq ID No:4) containing a HindIII site at the 5′ end, a ClaI site at the 3′ end,and a Kozak consensus (underlined) (Kozak M., Journal of BiologicalChemistry, 266:19867-19870 (1991)) for translation initiation aresynthesized on an ABI oligonucleotide synthesizer. Following gelpurification, the oligos are annealed and the ends of the fragmentphosphorylated using T4 polynucleotide kinase (NEB) and ATP. Theoligonucleotide pair ALM 1/2 was ligated to the linearizedpKK223/NY5IN-NdeI (D116N) and the product of the ligation reactiondigested with HindIII, to expose the HindIII site on the 5′ end (fromthe oligos), and to remove the entire IN coding sequence from thebacterial expression vector. The IN gene, optimized for mammaliantranslation, is isolated from the agarose gel. The sequence of thesynthetic gene so modified for translation in mammalian cells (Seq IDNo.: 1) is shown in FIG. 1. The mammalian expresion vector pRcRSV ispurchased from Invitrogen. Plasmid pRcRSV is digested with HindIII andthe ends dephosphorylated. Linearized plasmid is isolated from anagarose gel. The IN gene is ligated into the HindIII site of pRcRSV toproduce the plasmid pRSV/IN-NdeI (D116N). Correct orientation of theinsert is determined by restriction endonuclease digestion, and thesequence at the 5′ end of the gene (through the ClaI site) confirmed byDNA sequencing.

EXAMPLE 3

Demonstration of Transdominant Negative Activity of Integrase Expression

A vector expressing the synthetic gene of integrase is demonstrated tohave dominant negative activity against HIV infection by the followingexperiment. The integrase gene is transiently expressed in 293-CD4cells, which are subsequently infected by HIV. The cells expressing HIVintegrase support HIV replication substantially less than cells with nointegrase.

The 293-CD4 cells are split into 6-well plates at a cell density of2-4×10⁵ per well. Cells are allowed to attach and grow for 6 hours priorto transfections with the expression vector DNA. Following the protocolof Example 4 for 293 cell 10 transfections, the cells are incubated withcalcium phosphate precipitates for 24 hours. The medium on the cells isthen changed immediately prior to infection with HIV-1.

4-8×10⁴ Infectious HIV-IIIB particles are added i15 per well. Infectionis allowed to proceed for approximately 12 hours, and then the medium ischanged.

At various times after infection, samples of medium are removed forreverse transcriptase assay following the protocol of Example 5.

The following data is an example of the counts per minute obtained inthe reverse transcriptase assay from samples from such an assay:

Day PostInfection Vector pRSV/IN-NdeI Day 2 1054 1181 Day 3 1280  962Day 4 2682 1802 Day 5 5607 2792

This, and other experiments, establishes that expression of integrasecan substantially slow infection of HIV.

It should be noted that absolute blockage of viral replication cannot beexpected in a transient expression system, since not all of the cellsare expressing integrase.

EXAMPLE 4

Protocol for Transfection of 293 Cells

The transfection protocol has been modified for use specifically on 293and 293/CD4⁺cell lines. The same protocol is used for the expression andtransdominant experiments to introduce expression vectors. Transfectionefficiencies as high as 85-90% are routinely observed.

Solutions HBSS: Dextrose (6 mM) 1.19 g NaCl (137 mM) 8.01 g KCl (5 mM)0.37 g Na₂HPO₄ (0.7 mM) 0.10 g Hepes Na + (21 mM) 5.47 g dissolve in1000 mL ddH₂O pH solution to 7.05 with NaOH 2M CaCl₂: 29.4 g/100 mLddH₂O

Protocol

1. The 293 cell line is split out into appropriate tissue culturedishes.

Transdominant assay: 6-well plates

Expression study: 10 cm² dishes

The cells are allowed to adhere and spread for 6 hours prior totransfection.

2. Place 5 μg of vector DNA into 250 μL of HBSS (sufficient for one6-well or one 10 cm² dish).

Add 31 μ of 2M CaCl₂ and vortex gently for 1 to 2 minutes to thoroughlymix.

3. Incubate at room temperature for 45 minutes;

precipitate more than likely will not be visible.

4. Add the CaPO₄/DNA precipitate directly to a minimal amount of tissueculture media covering the cells.

6-well plate: 2 mL/well

10 cm² dish: 5 mL/dish

5. Incubate cells in the presence of precipitate overnight at 37° C. Donot glycerol shock or you will lose the majority of cells.

6. The next morning, aspirate off the old media and replace with newmedia. Then incubate for the necessary length of time for theexperiment.

EXAMPLE 5

Protocol for Reverse Transcriptase Assay

The reverse transcriptase (RT) assay measures the expression of viralproteins in the cultures.

RT reaction cocktail = 1.25 × RT reaction shock 50 mM Tris pH 8.3 10 mL1M 75 mM KCl 15 mL 1M 10 mM MgCl₂ 2 mL 1M 10 mM DTT 2 mL 1M 1 mM EGTA 76mg 0.5% NP-40 1 mL 10% 100 μg/mL poly rA 2 mL 10 mg/mL 25 μg/mL oligo dT128 mL dH₂O 100 A₂₆₀ units (1 bottle)

8 μCi/mL ³²P-dATP (400 Ci/mmol) is added immediately before assay.

Protocol

1. Place two GeNunc polypropylene modules (120 μL, Cat #2-32549) in aGeNunc frame (Cat #2-32042) for each assay plate.

2. Transfer 5 μL of culture media from each well of the assay plate tothe corresponding well of the GeNunc module. Using a 12-channelpipettor, transfer row H, change tips, and complete the rest of theplate starting at row A and moving to row G. By moving from the lowestto highest RT activity, the rest of the plate can be transferred withoutchanging tips.

3. Prepare 2.5 mL RT reaction cocktail for each plate by adding 2.5 μL³²P-dATP (400 Ci/mmol, 10 μCi/μL) to 2.5 mL 1.25×RT reaction stock.

4. Dispense 2.5 mL RT reaction cocktail into the trough of an 8-channelreagent reservoir for each assay plate. Use a P1,000 pipetman so thatthe radioactive tip can be discarded in the Ziplock waste bag.

5. Add 20 μL RT reaction cocktail to each well of the GeNunc module.Using a 12-channel pipettor, transfer row H, change tips, and finish therest of the plate stating at row A and moving to row G. By moving fromthe lowest to highest RT activity, the rest of the plate can be filledfrom one trough of the reagent reservoir without changing tips.

6. Cover the wells with GeNunc adhesive tapes (Cat #232700).

7. Incubate 2 hours, 37° C.

8. Mark a 96-well array on a sheet of Whatman DE81 anion exchange filterpaper using the rubber stamp.

9. Spot 4 μL of the RT reaction from each well of the GeNunc module ontothe corresponding mark of the DE81 filter. Using a 12-channel pipettor,transfer row H, change tips, and finish the rest of the module startingat row A and moving to row G. By moving from the lowest to highest RTactivity, the rest of the module can be spotted without changing tips.

10. Wash the filter five times, 3 minutes each, in 2×SSC (300 mM NaCl,30 mM NaCitrate, pH 7).

11. Rinse the filter twice in 95% ethanol.

12. Air dry the filter.

13. Quantitate the RT activity by counting the incorporated ³²p for eachwell using the Betagen Betascope 603 with the slot/dot blot analysisprogram as described in the applications manual.

Data is collected for 30 minutes in the ³²P mode and reported as totalcounts per well.

EXAMPLE 6

Construction of a CEM Cell Line Expressing HIV-1 Integrase

CEM cells are a line of CD4⁺human lymphoblastoid cells (obtainable fromAmerican Type Culture Collection). CEM cells were maintained in RPMI1640 medium supplemented with 10% fetal calf serum, 50 U/mL penicillin,50 U/mL streptomycin. All tissue culture reagents were obtained fromGibco BRL, Gaithersburg, MD. CEM cells were transfected byelectroporation according to the method of Aldovini and Feinberg (pp.147-159 in: Techniques in HIV Research, Stockton Press, New York, NY,1990). Twenty micrograms each of plasmid DNA (either pRc/RSV,pRSV/IN-Nde, or pRSV/IN-D116N) were added to a 0.4 mL suspension of 5million CEM cells in serum free RPMI 1640 medium. The DNA-cellsuspensions were incubated on ice for 10 minutes in a Gene Pulsarcuvette and then subjected to a single pulse of 960 μF at 300 voltsusing a Bio-Rad Gene Pulsar Electroporator (Bio-Rad, Richmond, Calif.Following electroporation, the cells were incubated on ice for 10minutes and then diluted in 10 mL RPMI 1640 medium with 10% fetal calfserum. The cells were incubated in 75 cm² tissue culture flasks at 37°C. in a 5% CO₂ incubator for 48 hours. The cells from each flask werecentrifuged to pellet the cells and the supernatants removed. The cellpellets were diluted in RPMI 1640 medium supplemented with 10% fetalcalf serum and 750 μg/mL G418 (Geneticin, Gibco BRL) at a density of200,000 cells per mL. The diluted cells were transferred to 96 wellplates, 100 μL/well, and incubated at 37° C. for 7 days. The G418selection was then increased to 1 mg/mL. Colonies appeared in 2-3 weeks.These colonies were transfered to 6 well plates and were diluted in 3 mLRPMI 1640 medium plus 1 mg/mL G418.

After the cells had reached a density of 1,000,000 cells per mL, theywere screened by western blot techniques for expression of HIV-1integrase, using a polyclonal rabbit antiserum prepared againstintegrase produced in E. coli (Holler T. P., et al., Gene, 136:323-328(1993)). The procedure used for this western blot is described. Onemillion cells were suspended in Laemmli buffer, and loaded onto a 12%polyacrylamide gel. The separated protein bands were transferred tonitrocellulose paper by electroblotting. The blots were blocked with 10%nonfat dry milk in phosphate buffered saline (PBS) plus 0.3% tween 80for 1 hour. The blocked blots were then incubated for 2 hours with a1/1000 dilution of the rabbit antiserum. The blots were then washed 3times in PBS/tween, then incubated for 1 hour with a 1/2000 dilution ofgoat antirabbit IgG conjugated with horseradish peroxidase. After 4washes in PBS-tween, the integrase expression was detected with anenhanced chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL).Of 50 wells which grew under G418 expression, two each expressing wildtype integrase or D116N were obtained.

EXAMPLE 7

Demonstration of Protection Against HIV Infection in CEM CellsExpressing a Dominant Negative Integrase Gene

CEM cell lines prepared in Example 6 were grown in the presence of 1mg/mL G418, and density adjusted to 4×10⁶ cells/mL. Fifty microliters ofsuspended cells was combined with 40 μL HIV stock virus (2×10³ pfu/mL)and 10 μL medium. Cells and virus were coincubated at 37° C. for 2hours, then washed once in 1 mL medium. Each washed pellet wasresuspended in 3 mL medium, then 1 mL aliquots were plated in triplicatewells of a 24 well plate. Fifty microliter samples were taken from eachwell of infected cells on days 1-5 and 7 postinfection. Cell cultureswere split 1:3 on days 3 and 5 after sampling, by adding 2 mL medium,mixing the culture, then removing 2 mL of medium and infected cells. Thedata is not corrected for these splits.

The samples were assayed using the reverse transcriptase assay describedin Example 5. The counts per minute from the triplicate samples at eachtime point were averaged.

Day Control Vector Nde D116N 1  598 2325 2035 2 1177 1701 1905 3 19471806 2260 4 6776 3102 3396 5 6815 2768 2935 7 22922  9820 7542

This experiment shows that expression of either the wild type integrase(Nde) or the D116N mutant retards the growth of HIV-1 in a humanlymphoblastoid cell line, as predicted by the results in the 293-CD4cells.

It should be noted that since these cells were not cloned beforetesting, even though all were G418 resistant, not all of them wereexpressing integrase. Therefore, the level of protection observed is aminimal level to be expected in a population expressing the dominantnegative integrase genes.

4 1 900 DNA Artificial Sequence Description of Artificial SequenceEncoding HIV Integrase 1 ccaagcttgg gccaccatgg ccttcctgga cggtatcgataaagctcagg aagaacacga 60 aaaataccac tctaactggc gcgccatggc ttctgacttcaacctgccgc cggttgttgc 120 caaggaaatc gtggcttctt gcgacaaatg ccaattgaaaggtgaagcta tgcatggtca 180 ggtcgactgc tctccaggta tctggcagct ggactgcactcatctcgagg gtaaagttat 240 cctggttgct gttcacgtgg cttccggata catcgaagctgaagttatcc cggctgaaac 300 cggtcaggaa actgcttact tcctgcttaa gctggccggccgttggccgg ttaaaactgt 360 tcacactgac aacggttcta acttcactag tactactgttaaagctgcat gctggtgggc 420 cggcatcaaa caggagttcg ggatcccgta caacccgcagtctcagggcg ttatcgaatc 480 tatgaacaaa gagctcaaaa aaatcattgg ccaggtacgtgatcaggctg agcacctgaa 540 aaccgcggtg cagatggctg ttttcatcca caacttcaaacgtaaaggtg gtatcggtgg 600 ttacagcgct ggtgaacgta tcgttgacat catcgctactgatatccaga ctaaagaact 660 gcagaaacag atcactaaaa tccagaactt ccgtgtatactaccgtgact ctagagaccc 720 ggtttggaaa ggtcctgcta aactcctgtg gaagggtgaaggtgctgttg ttatccagga 780 caactctgac atcaaagtgg taccgcgtcg taaagctaaaatcattcgcg actacggcaa 840 acagatggct ggtgacgact gcgttgctag ccgtcaggacgaagactaaa agcttcaggc 900 2 290 PRT Artificial Sequence Description ofArtificial Sequence HIV Integrase 2 Met Ala Phe Leu Asp Gly Ile Asp LysAla Gln Glu Glu His Glu Lys 1 5 10 15 Tyr His Ser Asn Trp Arg Ala MetAla Ser Asp Phe Asn Leu Pro Pro 20 25 30 Val Val Ala Lys Glu Ile Val AlaSer Cys Asp Lys Cys Gln Leu Lys 35 40 45 Gly Glu Ala Met His Gly Gln ValAsp Cys Ser Pro Gly Ile Trp Gln 50 55 60 Leu Asp Cys Thr His Leu Glu GlyLys Val Ile Leu Val Ala Val His 65 70 75 80 Val Ala Ser Gly Tyr Ile GluAla Glu Val Ile Pro Ala Glu Thr Gly 85 90 95 Gln Glu Thr Ala Tyr Phe LeuLeu Lys Leu Ala Gly Arg Trp Pro Val 100 105 110 Lys Thr Val His Thr AspAsn Gly Ser Asn Phe Thr Ser Thr Thr Val 115 120 125 Lys Ala Ala Cys TrpTrp Ala Gly Ile Lys Gln Glu Phe Gly Ile Pro 130 135 140 Tyr Asn Pro GlnSer Gln Gly Val Ile Glu Ser Met Asn Lys Glu Leu 145 150 155 160 Lys LysIle Ile Gly Gln Val Arg Asp Gln Ala Glu His Leu Lys Thr 165 170 175 AlaVal Gln Met Ala Val Phe Ile His Asn Phe Lys Arg Lys Gly Gly 180 185 190Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp Ile Ile Ala Thr 195 200205 Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Thr Lys Ile Gln Asn 210215 220 Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Val Trp Lys Gly Pro225 230 235 240 Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val Ile GlnAsp Asn 245 250 255 Ser Asp Ile Lys Val Val Pro Arg Arg Lys Ala Lys IleIle Arg Asp 260 265 270 Tyr Gly Lys Gln Met Ala Gly Asp Asp Cys Val AlaSer Arg Gln Asp 275 280 285 Glu Asp 290 3 35 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Oligonucleotides 3ccaagctggg ccaccatggc cttcctggac ggtat 35 4 38 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Oligonucleotides 4cgataccgtc caggaaggcc atggtggccc aagcttgg 38

What is claimed is:
 1. An isolated nucleic acid sequence comprising anucleic acid sequence encoding the polypeptide of SEQ ID NO:2.
 2. Avector comprising an isolated nucleic acid sequence encoding thepolypeptide of SEQ ID NO:2.
 3. The vector of claim 2, wherein thenucleic acid sequence comprises SEQ ID NO:1.
 4. The vector of claim 2,wherein the vector is selected from the group consisting of pRSV/IN-NdeIand pRSV/IN-D116N.
 5. The vector of claim 2, wherein the vector ispRSV/IN-NdeI.
 6. A method of making a vector encoding the polypeptide ofSEQ ID NO:2 comprising inserting an isolated nucleic acid sequenceencoding the polypeptide of SEQ ID NO:2 into a vector.
 7. The method ofmaking a vector of claim 6, wherein the nucleic acid sequence comprisesSEQ ID NO:1.
 8. The method of making a vector of claim 6, wherein thevector is selected from the group consisting of pRSV/IN-NdeI andpRSV/IN-D116N.
 9. The method of making a vector of claim 6, wherein thevector is pRSV/IN-NdeI.
 10. A method of transfecting a cell in vitrocomprising transfecting an isolated cell with a vector comprising anucleic acid sequence encoding the polypeptide of SEQ ID NO:2 such thatthe cell functionally expresses the polypeptide.
 11. The method oftransfecting a cell in vitro of claim 10, wherein the nucleic acidsequence comprises SEQ ID NO:1.
 12. The method of transfecting a cell invitro of claim 10, wherein the vector comprising a nucleic acid sequenceencoding SEQ ID NO:2 is selected from the group consisting ofpRSV/IN-NdeI and pRSV/IN-D116N.
 13. The method of transfecting a cell invitro of claim 12, wherein the vector comprising a nucleic acid sequenceencoding SEQ ID NO:2 is pRSV/IN-NdeI.
 14. The method of transfecting acell in vitro of claim 10, wherein the cell is a mammalian cell or anavian cell.
 15. The method of transfecting a cell in vitro of claim 14,wherein the mammalian cell is 293-CD4.
 16. The method of transfecting acell in vitro of claim 14, wherein the mammalian cell is a human cell.17. The method of transfecting a cell in vitro of claim 16, wherein thehuman cell is a CD4+ T lymphocyte or a monocyte.
 18. The method oftransfecting a cell in vitro of claim 16, wherein the human cell ishematopoietic progenitor cell.
 19. The method of transfecting a cell invitro of claim 10, wherein the cell is transfected using calciumphosphate co-precipitation, a complex of cationic liposomes,electroporation, naked DNA, or a viral vector.
 20. The method oftransfecting a cell in vitro of claim 19, wherein the viral vector isselected from the group consisting of a retroviral vector; an adenoviralvector; an adeno-associated viral vector; and a herpes viral vector. 21.A method of making the retroviral integrase of SEQ ID NO:2 comprising:a) making a vector encoding the polypeptide of SEQ ID NO:2 comprisinginserting an isolated nucleic acid sequence encoding the polypeptide ofSEQ ID NO:2 into a vector; b) transfecting an isolated cell with avector comprising a nucleic acid sequence encoding the polypeptide ofSEQ ID NO:2 such that the cell functionally expresses the polypeptide;and c) isolating the polypeptide of SEQ ID NO:2.
 22. The method ofmaking the retroviral integrase of SEQ ID NO:2 of claim 21, wherein thenucleic acid sequence comprises SEQ ID NO:1.